Metabolic and Transcriptomic Profiling Reveals Etiolated Mechanism in Huangyu Tea (Camellia sinensis) Leaves

Abstract

Leaf color is one of the key factors involved in determining the processing suitability of tea. It relates to differential accumulation of flavor compounds due to the different metabolic mechanisms. In recent years, photosensitive etiolation or albefaction is an interesting direction in tea research field. However, the molecular mechanism of color formation remains unclear since albino or etiolated mutants have different genetic backgrounds. In this study, wide-target metabolomic and transcriptomic analyses were used to reveal the biological mechanism of leaf etiolation for 'Huangyu', a bud mutant of 'Yinghong 9'. The results indicated that the reduction in the content of chlorophyll and the ratio of chlorophyll to carotenoids might be the biochemical reasons for the etiolation of 'Huangyu' tea leaves, while the content of zeaxanthin was significantly higher. The differentially expressed genes (DEGs) involved in chlorophyll and chloroplast biogenesis were the biomolecular reasons for the formation of green or yellow color in tea leaves. In addition, our results also revealed that the changes of DEGs involved in light-induced proteins and circadian rhythm promoted the adaptation of etiolated tea leaves to light stress. Variant colors of tea leaves indicated different directions in metabolic flux and accumulation of flavor compounds.

Keywords: Camellia sinensis cv. Huangyu; biological mechanism; chlorophyll; leaf color; yellowing.

Figure 1

Phenotypic characteristics of Yinghong 9 (left) and Huangyu (right) (A) and pigment contents (B–D) of ‘Yinghong 9’ and ‘Huangyu’. The data are presented as the mean ± standard deviation (n = 3). * indicates significant difference (p < 0.05) between ‘Yinghong 9’ and ‘Huangyu’. YJ is ‘Yinghong 9’, HY is ‘Huangyu’.

Figure 2

Metabolome quality and differential metabolites analysis: (A) OPLS-DA, (B) Permutation test for OPLS-DA model, Q² > 0.5, R² > 0.5, p < 0.005; (C) Type of differential metabolites and expression of up-/down-regulation; and (D) Differential metabolites of Top 20 FC (fold change of YJ/HY) between ‘Yinghong 9’ (YJ) and ‘Huangyu’ (HY)). YJ was ‘Yinghong 9’ leaves, HY was ‘Huangyu’ leaves. pmn001519, Galloyl Methyl gallate; Lmfn001258, Maplexin D; Zmhn005413, m/p Dimeric galloyl methyl ester; Lmsn002071, Tellimagrandin I; pme2246, Ellagic acid; mws1293, Theaflavin; Wmhn004451, 1,4,8-Trihydroxynaphthalene-1-O-[6′-O-(3′,4′,5′- trimethylbenzoyl)] glucoside; pmn001367, Protocatechuic acid-4-glucoside; pmb2871, 2,5-Dihydroxy benzoic acid O-hexside; pmn001382, Isochlorogenic acid A; pmn001628, Hexahydroxydiphenoylglucose; Li512115, 3,4-Dicaffeoylquinic acid; pmn001525, 3,5-Di-O-galloylshikimic acid; Zmlp007578, 3,3′,4-Trimethoxy ellagic acid; pmp001285, Phthalic anhydride; Zmhn002783, 5-O-Galloylshikimic acid; pmn001516, 5-Galloylshikimic acid; pmn001384, Isochlorogenic acid C; pme1261, Pantothenol; mws0473, 2-Methylsuccinic acid; pme3011, γ-Aminobutyric acid; mws0574, 2-Hydroxyisobutyric acid; mws1060, 9-(Arabinofuranosyl)hypoxanthine; pmb2789, 13S-Hydroperoxy-6Z,9Z,11E-octadecatrienoic acid; Lmxp010913, 1-(9Z-Octadecenoyl)-2- (9-oxo-nonanoyl)-sn-glycero-3-phosphocholine; pmb2791, 9-HpOTrE; pmb1650; Octadeca-11E,13E,15Z-trienoic acid; Lmhp008440, LysoPE 15:1; mws0289, LysoPE 18:1; pmb0885, 4-O-9Z,11Z,13E,15E-octadecatetraenoic acid; Lmhp009773, 1-α-Linolenoyl- glycerol-glucoside; pmb0128, δ-Tridecalactone; pmn001691, 9,12,13-Trihyroxy-10,15-octadecadienoic acid; mws1012, Coumarin; pmb0631, Luteolin-8-C-glucoside-6-C-arabinoside; pmb0624,Luteolin-6-C-glucoside-7-O-glucoside; pmp000786, Eupatorin; pmb0636, Luteolin-8-C-glucoside-7-O-arabinoside; mws1608, Luteolin-6-C-glucoside (Isoorientin); pme1540, Isorhamnetin-3-O-neohesperidoside; Xmyp004945, Camelliaside A; Lmmp002755, Quercetin-7-O-rutinoside-4′-O-glucoside; mws0636, Phe-Phe; pmb2211, Cocamidopropyl betaine; pmp001280, 3-{[(2-Aminoethoxy)(hydroxy)phosphoryl] oxy}-2-hydroxypropyl-9,12-octadecenoate; pme2268, Trigonelline.

Figure 3

Heat map of carotenoids in ‘Yinghong 9’ and ‘Huangyu’. Red and blue indicate higher and lower abundances, respectively. YJ was ‘Yinghong 9’; HY was ‘Huangyu’.

Figure 4

Volcano plot of differentially expressed transcripts (A) and KEGG enrichment analysis of DEGs (B) in ‘Yinghong 9’ and ‘Huangyu’.

Figure 5

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of the DEGs. (A) Relative expression levels of genes. * indicates significant difference (p < 0.05) between ‘Yinghong 9’ and ‘Huangyu’. (B) Correlation analysis between the FPKM value and qRT-PCR results. All data are shown as mean ± SE (n = 3). YJ was ‘Yinghong 9’ fresh leaves, HY was ‘Huangyu’ fresh leaves.

Figure 6

Expression profiles of DEGs involved in chlorophyll metabolism (A) and photosynthesis (B) photosynthesis-antenna proteins and photosynthetic proteins involved in endoplasmic reticulum pathway in ‘Yinghong 9’ and ‘Huangyu’. YJ is ‘Yinghong 9’, HY is ‘Huangyu’.

Figure 7

DEGs involved in flavonoid pathway and expression levels in ‘Yinghong 9’ and ‘Huangyu’. The heatmap is created according to the average expression levels of related biosynthetic genes based on the FPKM value by RNA-seq. Green indicates low expression levels, and red indicates high expression levels. ANS, anthocyanidin synthase; DFR, dihydroflavonol reductase; F3′5′H, flavonoid 3′,5′-hydroxylase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase. Heavy lines indicate the enhancement of metabolic flux in ‘Huangyu’, and thin lines indicate the attenuation of metabolic flux in ‘Yinghong 9’.